The Prokaryotic Expression Of Human Pregnancy Specific β1Glycoprotein And Preparation Of Its Antibodits

PSG1is a kind of specific glycoprotein which is synthesized and secreted into theblood by the placental syncytiotrophoblast cells.The changes in the expression ofPSG1protein have indicativefunction to early pregnancy,placental function, and otherpregnancy abnormalities.When fetal growth and survival in the disadvantageousintrauterine environment, the expression of PSG1is relatively reduced.Now thatdetermination of the concentration of maternal blood PSG1can determine placentalfunction, reflecting the health of syncytiotrophoblast layer of cells.It is a trustedreference index, it also have implications for clinical prenatal screening and diagnosisof related disease.PSG1gene is located on chromosome19q13.2, the full-length of its mRDNA is2306bp.It encodes426amino acids, of the mature peptide part of encoding392aminoacid residues, Prokaryotic expression molecular weight of about45kDa.At present,thedetection of PSG1during pregnancy has gradually been clinician’s attention.and it hasbeen widely used in pregnant women prenatal health checks and diagnostic relateddiseases.Therefore, the development of immunological test kit has broad clinicalapplication.In this study, we obtain PSG1protein by recombinant genetic engineeringtechnology and affinity purification technology.PSG1polyclonal antibodies andmonoclonal antibodies had been gotten with animal immunization and hybridomatechnology. Provide a theoretical basis for the PSG1protein in the development andtreatment of clinically relevant disease prenatal placental function test kit.1. Gene cloning of PSG1, prokaryotic expression and purificationThe primer of PSG1gene has been design according to the mRNA sequence ofthe human GenBank PSG1gene （NM₀06905.2）.Then the PSG1gene was amplifiedby RT-PCR.The resulting PCR product was inserted into the plasmid vector pGEM-Tto build pCzn1-PSG1expression vector after sequencing is correct.Transformed itinto E. coli ArcticExpressTM （DE3） with IPTG induction for efficient expression.Theexpression product was purified using affinity chromatography etc, extraction and purified to get a protein which molecular weight is approximately45kDa.The purityof PSG1protein is not less than90%.2. Preparation and purification of Rabbit anti-PSG1polyclonal antibodyNew Zealand white rabbits were immunized with PSG1protein asimmunogens.Determine the titer of antiserum against immunogenic protein is greaterthan1:5.0×104by indirect ELISA after four times immunization.Blood was collectedfrom carotidartery, andantiserum was prepared.And the antibody was purified by theantigen specificity and affinity chromatography.The purified antibody concentrationwas0.33mg/mL, more than90%purity,the titer of antiserum against immunogenicprotein is greater than1:1.28×105by indirect ELISA. The polyclonal antibody ofPSG1protein was successfully prepared with high specificity and high titer.3. Preparation and purification of Mouse anti-PSG1monoclonal antibodiesInject the mouse with1mg/mL PSG1recombinant protein by intraperitonealinjection,50μg each BALB/c mice.Determine the titer of antiserum againstimmunogenic protein is greater than1:5.0×104by indirect ELISA after four timesimmunization.Carried hybridoma fusion cloned, antibody screening and massproduction of ascites.And the antibody was purified by the antigen specificity andaffinity chromatography.The purified antibody concentration was0.8mg/mL, morethan95%purity,the titer of antiserum against immunogenic protein is greater than1:2.43×105by indirect ELISA.The polyclonal antibody of PSG1protein wassuccessfully prepared with high specificity and high titer.In summary,This study successfully constructed pCzn1-PSG1expressionvector,then transferred it into the matched E.coli ArcticExpressTM （DE3） to obtainedthe good immunogenicity and specificity PSG1protein. Further successfully preparedhigh titer PSG1specific polyclonal and monoclonal antibodies.Lay the foundation forthe development of PSG1protein immunoassay kit.